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Abstract #20381 Published in IGR 10-1
Distinct P2Y receptor subtypes regulate calcium signaling in human retinal pigment epithelial cells
Tovell VE; Sanderson JInvestigative Ophthalmology and Visual Science 2008; 49: 350-357
PURPOSE: Nucleotide signaling plays a role in retinal pigment epithelial (RPE) function, and receptors for nucleotides are potential therapeutic targets for various ocular diseases. The purpose of this study was to investigate the expression of P2Y receptor subtypes in native and cultured human RPE cells. METHODS: Intracellular Ca2+ levels were monitored using real-time fluorescence imaging in cultured human RPE cells loaded with Fura-2. Expression of P2Y receptors in native and cultured RPE cells was determined by quantitative RT-PCR and Western blot analysis. RESULTS: Adenosine triphosphate (ATP), uridine triphosphate (UTP), adenosine diphosphate (ADP), 2-methylthio ATP (2MeSATP), and uridine diphosphate (UDP) produced concentration-related increases in [Ca2+]i in cultured RPE cells. However, differences between the magnitude and shape of agonist responses were observed. ATP and UTP showed similar response characteristics, including a distinct Ca2+ influx component. ATP and UTP were equipotent (EC50, 6 μM) and maximum responses were equivalent, suggesting activation of a P2Y2 receptor. Maximal responses to ADP and 2MeSATP were equivalent with EC50s of 1 μM and 0.3 μM. The P2Y1 antagonist MRS 2179 (10 μM) inhibited these responses, confirming functional expression of P2Y1 receptors. The presence of a response to UDP suggested P2Y6 expression. There was no influx component to P2Y1- and P2Y6-mediated responses. mRNA for P2Y1, P2Y2, P2Y4, and P2Y6 receptor subtypes was found in cultured RPE cells, and for P2Y1, P2Y2, P2Y4, P2Y6, and P2Y(12) it was found in native RPE cells. Expression of P2Y1, P2Y2, and P2Y6 protein was found in native and cultured RPE cells. CONCLUSIONS: These data define the expression profile of P2Y receptors in human RPE and show that different P2Y subtypes control distinct calcium responses in these cells.
Dr. V.E. Tovell, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, United Kingdom
Classification:
2.13 Retina and retinal nerve fibre layer (Part of: 2 Anatomical structures in glaucoma)
3.6 Cellular biology (Part of: 3 Laboratory methods)
3.5 Molecular biology incl. SiRNA (Part of: 3 Laboratory methods)
